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Definitions

• the disclosed processes, methods, and systems are directed to treating a subject with high levels of cholesterol or at risk thereof by enhancing cholesterol shuttling into and degradation within the mitochondria.
• High cholesterol is the presence of high levels of cholesterol in the blood that may be the result of poor diet, lifestyle, disease (e.g. diabetes) or genetics. High concentrations of cholesterol within the cell is a characteristic of several diseases, for example fatty liver disease, atherosclerosis, etc.
• Atherosclerosis involves narrowing of arteries resulting from a buildup of cholesterol-laden lipoproteins within the arterial wall. Often a precursor to leading causes of death including cardiovascular disease (CVD), myocardial infarction, stroke and peripheral vascular disease.
• CVD cardiovascular disease
• myocardial infarction myocardial infarction
• stroke peripheral vascular disease.
• LDLs low density lipoproteins
• HDLs high-density lipoproteins
• compositions and methods for reducing lipid and cholesterol levels in subjects suffering from excess lipid and cholesterol are needed.
• compositions, methods, and systems for enhancing transport of cholesterol (including various modified cholesterol compounds) into modified mammalian mitochondria, wherein the modification renders the mitochondria capable of catabolizing cholesterol may be engineered for enhanced cholesterol flow across the mitochondrial membrane.
• the cells may also include one or more proteins that may aid in transport of cholesterol across the mitochondrial membrane.
• the disclosed compositions may be administered to a subject in need of treatment for high cholesterol.
• the nucleic acids expressing the disclosed proteins may be incorporated into a mammalian cell’s genome or may be extra-genomic.
• the disclosed cells may be selected from a liver cell, neuronal cell, bone cell, muscle cell, endothelial cell, epithelial cell, immune cell, hematopoietic cell, and stem cell or precursor cell thereof.
• mammalian mitochondria are engineered to comprise one or more recombinant and/or non-endogenous proteins that may help enhance cholesterol flow across the mitochondrial membrane.
• the mitochondria may also include one or more proteins that may aid in catabolism of cholesterol.
• the disclosed modified mitochondria may be useful in reducing intracellular cholesterol concentrations, increasing cholesterol transport across the mitochondrial membrane, and/or reducing concentrations of cholesterol in the blood of a patient or subject at risk of developing, or suffering from high cholesterol or hypercholesterolemia.
• a method of modifying a mammalian mitochondrion includes inserting at least one non-native engineered Steroidogenic Acute Regulatory (StAR) protein or portion thereof into the mammalian mitochondrion outer membrane, and where the mitochondrion includes one or more of a cholesterol degrading protein (CDP) includes cholesterol dehydrogenase (CholD), 3-ketosteroid A1 -dehydrogenase (A1-KstD), anoxic cholesterol metabolism B enzyme (acmB), 3-ketosteroid 9a- hydroxylase (KshAB), 3
• CDP cholesterol dehydrogenase
• A1-KstD 3-ketosteroid A1 -dehydrogenase
• acmB anoxic cholesterol metabolism B enzyme
• the StAR protein may comprise residues 63 to 188 of SEQ ID NO:7, a duplication thereof, or at least a portion of the TOMM20 protein.
• the CDP may comprise P450 or P450- ferredoxin reductase-ferredoxin fusion protein (P450-FdxR-Fdx).
• the StAR protein and the CDP may comprise at least one mitochondrial signaling protein and/or be expressed from a cell-specific promoter sequence, and/or be expressed from a genome-integrated or from an extra genomic gene, and/or at least about 10% of the total amount of StAR protein and CDP in the cell may be located in a mitochondrial membrane.
• the disclosed methods and compositions may be useful in treating a subject suffering from or at risk of developing high cholesterol, such as by reducing cholesterol in the subject having high cholesterol.
• the cell may be selected from a liver cell, neuronal cell, bone cell, muscle cell, endothelial cell, epithelial cell, immune cell, hematopoietic cell, and stem cell or precursor cell thereof.
• FIG. 3 bar chart showing cholesterol degradation and consequent production of catabolite is greatly increased in vitro by the expression of StAR in mouse RAW264.7 cell lines expressing CDP.
• FIG. 4 is a Western blot showing protein amounts in cell lines described in FIG. 3.
• FIG. 5 are sequences of various proteins and nucleic acid coding sequences disclosed herein. Specifically, the Human protein sequence for p450-FdxR-Fdx (SEQ ID NO:1) is depicted with the Flag tag in bold; Cholesterol dehydrogenase, CholD (SEQ ID NO:2), is shown; n3-ketosteroid A1 -dehydrogenase, A1-KstD (SEQ ID NO:3), is shown; Anoxic cholesterol metabolism B enzyme, acmB (SEQ ID NO:4) is shown; 3-ketosteroid 9a- hydroxylase, KshAB (SEQ ID NO:5) is shown; 3
• Disclosed herein is the development of a unique approach to help manage hypercholesterolemia and other disorders related to the presence of excessive cholesterol. Humans lack enzymes to degrade cholesterol and many mammalian cells take up excess cholesterol without a way to properly store or metabolize it. In some cases, macrophages progress to foam cells as they fill with cholesterol and cholesterol esters. This buildup of cholesterol may lead to formation of arterial plaques that can lead to atherosclerosis.
• plaques may cause coronary artery disease, angina (chest pain), intermittent claudication (leg pain with exercise) or chronic kidney disease due to poor circulation.
• many plaques can be unstable and prone to rupture even before they have any significant effect on blood flow. These structures are typically clinically silent until they rupture (complicated plaques) and predispose the underlying tissue to clots which may suddenly occlude the affected blood vessel.
• human cells may be engineered to aid in reducing cholesterol.
• the cells may be any mammalian cell.
• the disclosed cells may be engineered to express various proteins that aid in transport and degradation/catabolism of cholesterol.
• an engineered cell may be a cell with one or more non-native and/or exogenous coding sequences or protein, for example an engineered mammalian cell may include one or more mammalian and/or bacteria-related sequences or proteins.
• Non-native, exogenous, or non-endogenous, as used herein may refer to a sequence or protein that is introduced to the cell, in most cases by recombinant methods.
• bacteria-related may refer to a protein or coding sequence with greater than about 50% identity with a similar bacterial sequence.
• the disclosed proteins may be fusion proteins and/or modified proteins, for example a protein lacking a sequence found in native proteins and/or duplicating sequences found in native proteins.
• the disclosed proteins may be over-expressed relative to endogenous genes and/or proteins.
• an endogenous gene or protein may be mammalian p450scc, StAR, or TOMM20 protein.
• over-expression of the disclosed proteins may result in 2X, 3X, 4X, 5X, 10X, 20X, 100X, 200X, based on weight or moles, or more of the disclosed gene or protein in a given sample compared to the endogenous gene or protein.
• overexpression is relative to noninduced expression of the endogenous protein.
• over-expression may be relative to induced levels of endogenous protein, wherein expression may be similar or the same.
• degradation in reference to degradation of cholesterol refers to removal of various side chains and/or opening of at least one ring of cholesterol.
• the disclosed coding sequences and proteins for cholesterol degradation may be derived from bacteria and/or mammals.
• the disclosed proteins may be referred to as cholesterol degrading proteins (CDP) and may be selected from one or more of cholesterol dehydrogenase (CholD; SEQ ID NO:2), 3- ketosteroid A1 -dehydrogenase (A1-KstD; SEQ ID NO:3), anoxic cholesterol metabolism B enzyme (acmB; SEQ ID NO:4), 3-ketosteroid 9a-hydroxylase (KshAB; SEQ ID NO:5), 3
• CDP cholesterol de
• the disclosed coding sequences and proteins may be delivered to the cell in- vivo or in-vitro, for example by various methods including electroporation, transfection, viral vector, nanoparticle, etc.
• the disclosed coding sequences and proteins may be encoded by one or more nucleic acids within a cell, vector, or particle.
• the particle may be lipid nanoparticle (or lipo nanoparticle; LNP).
• the vector may be viral vector, such as adeno virus or lentivirus.
• the coding sequences may include one or more promoter and/or enhancer sequences that may aid in expressing the coded proteins in a specific cell or tissue, or may aid in supporting high expression of the coded proteins, generally, or in a specific cell or tissue.
• CDPs cholesterol degrading proteins
• the disclosed proteins may be directed to the mitochondria of a mammal, for example the outer membrane, inner membrane, intermembrane space, or matrix.
• CDPs cholesterol degrading proteins
• the term “about” or “approximately” means an acceptable error for a particular value as determined by one of ordinary skill in the art, which depends in part on how the value is measured or determined. In certain embodiments, the term “about” or “approximately” means within 1 , 2, 3, or 4 standard deviations.
• the term “about” or “approximately” means within 30%, 25%, 20%, 15%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, or 0.05% of a given value or range. Whenever the term “about” or “approximately” precedes the first numerical value in a series of two or more numerical values, it is understood that the term “about” or “approximately” applies to each one of the numerical values in that series.
• administration may be “intravenous” administration, referring to introducing the compositions, constructs, and/or cells into a vein of a patient, e.g. by infusion (slow therapeutic introduction into the vein).
• administration may be “subcutaneous” wherein introduction is beneath the skin of the patient.
• “Infusion” or “infusing” refers to the introduction with a solution into the body through a vein for therapeutic purposes. Generally, this is achieved via an intravenous (IV) bag, such as a bag that can hold a solution.
• IV intravenous
• compositions, constructs, cells, methods, and systems may be “co-administered” to a patient, for example by intravenously administering at least a second therapeutic agent during the same administration.
• coadministration is concurrent, in others co-administration is sequential.
• prevention means the avoidance of the occurrence or of the re-occurrence of a disease as specified herein, or at least one symptom associated therewith, by the administration of a composition, construct, method, or system according to the invention to a subject in need thereof.
• a “patient” or “subject” includes various animals including a human, monkey, cat, dog, mouse, rat, rabbit or guinea pig.
• the animal can be a mammal such as a nonprimate and a primate (e.g., monkey and human).
• a patient is a human, such as a human infant, child, adolescent or adult.
• treat refers to eliminating, reducing, suppressing, or ameliorating, either temporarily or permanently, either partially or completely, at least one symptom, manifestation, or progression of an event, disease, disorder, or condition disclosed herein.
• methods, compositions, and systems employed as therapies may reduce the severity of a given disease state, but need not abolish every symptom associated therewith to be regarded as useful.
• a prophylactically administered treatment need not be completely effective in preventing the onset of a condition to constitute a viable prophylactic composition, agent, method or system.
• a disease for example, as disclosed herein, reducing blood cholesterol concentrations and/or reducing the number or severity of associated symptoms, or by increasing the effectiveness of another treatment, or by producing another beneficial effect), or reducing the likelihood that the disease will occur or worsen in a subject, is sufficient.
• the term “effective amount” refers to an amount of a compound of the invention or other active ingredient sufficient to provide a therapeutic or prophylactic benefit in the treatment or prevention of a disease or to delay or minimize symptoms associated with a disease.
• a therapeutically effective amount with respect to a compound of the invention means that amount of therapeutic agent alone, or in combination with other therapies, that provides a therapeutic benefit in the treatment or prevention of a disease. Used in connection with a compound of the invention, the term can encompass an amount that improves overall therapy, reduces or avoids symptoms or causes of disease, or enhances the therapeutic efficacy or synergies with another therapeutic agent.
• terapéuticaally effective amount means an amount of a composition of the present invention that (i) treats the particular disease, condition, or disorder, (ii) ameliorates or eliminates one or more symptoms of the particular disease, condition, or disorder, or (iii) prevents or delays the onset of one or more symptoms of the particular disease, condition, or disorder described herein.
• the therapeutically effective amount of the disclosed compositions may reduce the concentration of cholesterol in the patient’s blood; reduce intercellular cholesterol; inhibit (i.e., slow to some extent and preferably stop) cholesterol plaque growth and/or development; inhibit (i.e., slow to some extent and preferably stop) lipid buildup in the liver; reduce lipid content in the liver; and/or relieve to some extent one or more of the symptom associated with the high concentrations of blood cholesterol.
• the term “amelioration” as used herein refers to any improvement of a disease state (for example high blood cholesterol) of a patient, by the administration of one or more treatments and/or compositions, according to the present disclosure, to such patient or subject in need thereof.
• Such an improvement may be seen as a slowing down or stopping the progression of the disease of the patient, and/or decreasing the severity of at least one disease symptom, an increase in frequency or duration of disease symptom-free periods or a prevention of impairment or disability due to the disease.
• the cells may be endothelial and epithelial cells in all organs and tissues, including, without limitation, smooth, skeletal and cardiac muscle cells, neuronal cells, glandular cells, bone cells, immune cells, hematopoietic cells, and stem cells or precursor cells thereof.
• the cell maybe a pluripotent stem cells, for example an induced pluripotent stem cell.
• the tissues may be cancerous or pre-cancerous, and may one or more cells of skin and intestinal carcinomas (squamous epithelial cells), adenocarcinoma (epithelial cells of glandular origin, pancreatic) gastrointestinal carcinoid (neuroendocrine ceils), pancreatic cancer, small cell carcinoma (neuroendocrine cells), leiomyosarcoma (smooth muscle cells) and lymphomas (lymphocytes found in organs/walls of the gastrointestinal tract).
• skin and intestinal carcinomas squamous epithelial cells
• adenocarcinoma epidermal cells
• gastrointestinal carcinoid neuroendocrine ceils
• pancreatic cancer small cell carcinoma
• leiomyosarcoma smooth muscle cells
• lymphomas lymphomas
• LNPs may include one or more molecules that help target specific cells, for example a protein with affinity for a receptor or membrane protein on the target cell.
• the disclosed genes and proteins may aid in transport and/or metabolism of cholesterol.
• the disclosed genes and proteins may be expressed in and/or targeted to the mitochondria.
• the disclosed genes and proteins may allow cells to degrade and/or catabolize cholesterol to ring opening, whereupon endogenous proteins and enzymes may further metabolize the molecule.
• Mitochondria are found in most eukaryotic cells and organisms, and consist of a double membrane. Mitochondria supply the cell’s energy, and play a role in signaling, cell-cycle, cell growth and differentiation, apoptosis, and cell death. Cells with high demand for energy, such as heart, muscle, brain, and liver cells, may have several thousand mitochondria.
• the double-membrane structure of the mitochondria allows for specialized compartments: the outer membrane, the intermembrane space, the inner membrane, and the cristae and matrix. These compartments allow for specialized functions - such as ATP synthesis.
• the matrix is home to the mitochondrion’s independent genome.
• Steroids and steroidal hormones are synthesized from cholesterol.
• the first step in steroidogenesis is the cleavage of cholesterol’s side chain by P450scc, which is located on the matrix side of the inner mitochondrial membrane (IMM).
• IMM inner mitochondrial membrane
• cells that produce steroid hormones store and possess only small amounts of hormone at any one time.
• increases in steroid secretion is accomplished by first increasing synthesis of the steroid.
• steroids have broad and powerful action within the cell and organism - thus steroidogenic cells tightly regulate production of steroids and precursors to avoid overproduction and pathologies associated with excess amounts of steroid. Synthesis can be rapidly inducted and rapidly terminated to avoid overproduction.
• One way cells exert this control (which can increase synthesis 10-100- fold within minutes) is to regulate the flow of cholesterol into the mitochondrial matrix. Cholesterol is transported across the mitochondrial membrane by the StAR protein.
• the StAR protein is synthesized as a 37 kDa protein comprising a mitochondrial targeting leader peptide sequence. Upon insertion into the mitochondrial membrane, the leader peptide is cleaved to yield the 30 kDa protein that is located intra- mitochondrially. Mutations in the StAR gene can result in congenital lipoid adrenal hyperplasia. Subjects suffering from adrenal hyperplasia synthesize very small amounts of steroid.
• StAR may interact with two additional proteins to support importation of cholesterol into mitochondria.
• TSPO Translocator Protein
• TSPO expression is upregulated in response to exposure of macrophages to modified LDLs.
• PBR peripheral-type benzodiazepine receptor
• PBR peripheral-type benzodiazepine receptor
• non-steroidogenic cells i.e. cells that do not typically produce steroids or produce very low amounts of steroids
• StAR adrenodoxin reductase-adrenodoxin-COOH
• P450scc adrenodoxin reductase-adrenodoxin-COOH
• StAR adrenodoxin reductase-adrenodoxin-COOH
• StAR-StAR protein is a fusion protein that combines 1-188 residues of StAR to 63-285 residues of StAR, thus dimerizing the protease-resistant domain of StAR (residues 63-188).
• StAR proteins for use with the present compositions and methods may include various forms of the StAR protein sequence.
• the disclosed StAR proteins may include one or more mutations, truncations, deletions, duplications, fusions, etc. or the disclosed proteins may be wild-type or native StAR proteins.
• the disclosed StAR proteins may be modified to reduce transit time through the outer membrane, which may help to enhance mitochondrial import of cholesterol.
• cholesterol refers to cholesterin or cholesteryl alcohol, a sterol of formula C27H46O, with IUPAC names cholest-5-en-3
• cholesterol may also refer to derivatives of cholesterol, including oxidized cholesterol, C27H46O2, Oxycholesterol, or 5,6-epoxycholesterol, 7- ketocholesterol (7KC), cholestane-3
• the disclosed genes and proteins that may degrade cholesterol may be selected from cholesterol dehydrogenase (CholD), 3-ketosteroid A1 -dehydrogenase (A1-KstD), anoxic cholesterol metabolism B enzyme (acmB), 3-ketosteroid 9a- hydroxylase (KshAB), 3
• CholD cholesterol dehydrogenase
• A1-KstD 3-ketosteroid A1 -dehydrogenase
• acmB anoxic cholesterol metabolism B enzyme
• KshAB 3-ketosteroid 9a- hydroxylase
• HSD2 3
• One or more of the disclosed genes, proteins, and enzymes may be packaged into one or more vector, construct, or cassette.
• a cassette that includes one or more cholesterol degrading enzymes may be referred to as a cholesterol catabolizing cassette (CCC).
• the cassette may be a construct and may include a nucleic acid sequence that codes for at least one protein that is about 80% or more identical to a protein disclosed herein or a protein coded for by any of the disclosed genes.
• the percent identity with the disclosed protein or nucleotide sequences may be greater than about 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, and less than about 100%, 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 89%, 88%, 87%, 86%, 85%, 84%, 83%, 82%, and 81%.
• Sequence identity may be calculated for a protein sequence of greater than about 40 aa (amino acid residues), 50 aa, 60 aa, 70 aa, 80 aa, 90 aa, 100 aa, 110 aa, 120 aa, 130 aa, 140 aa, 150 aa, 160 aa, 170 aa, 180 aa, 190 aa, 200 aa or more and less than about 300 aa, 200 aa, 190 aa, 180 aa, 170 aa, 160 aa, 150 aa, 140 aa, 130 aa, 120 aa, 110 aa, 100 aa, 90 aa, 80 aa, 70 aa, or 60 aa.
• sequence identity is calculated over about 80-150 amino acid residues. In most cases an inserted or deleted amino acid, compared to the reference sequence (i.e. SEQ ID NOs: 1-10), counts as a single non-identical amino acid.
• the composition may include one or more cholesterol degrading proteins, for example an enzyme(s) and a cholesterol transporting protein, for example StAR protein or StAR variant.
• a cholesterol transporting protein for example StAR protein or StAR variant.
• the therapeutic cell may be administered by one or more vectors, constructs, and/or cassettes comprising nucleic acids that code for a cholesterol degrading protein, for example an enzyme and a cholesterol transport protein, for example StAR or a StAR variants.
• the nucleic acids may be transduced into the cell, for example by one or more viral vectors (in one example lentivirus) to aid in integrating or inserting the coding sequence into the genomic sequence or mitochondrial sequence of the cell.
• the nucleic acids may not be integrated, and the disclosed proteins are expressed from extra-genomic sequences in the nucleus or mitochondria.
• a subject or patient at risk for, or suffering from, high cholesterol may have total cholesterol concentration of greater than about 200 mg of cholesterol (i.e. low-density lipoprotein + high-density lipoprotein) per decilitre of blood and/or more than about 5.2 mmol/L total cholesterol.
• a subject or patient at risk for, or suffering from, high cholesterol may have an LDL concentration of greater than about 100 mg/dl and/or more than about 2.6 mM LDL.
• a subject or patient at risk for, or suffering from, high cholesterol may have an HDL concentration of less than about 50 mg/dl and/or less than about 1 .3 mM HDL.
• a subject or patient at risk for, or suffering from, high cholesterol may have a triglyceride concentration of greater than about 150 mg/dl and/or greater than about 1.7 mM triglycerides.
• a subject or patient at risk for, or suffering from, high cholesterol may have an LDLHDL ratio of about 4.0 (i.e. 4 to 1) or more than 4.0.
• Acetylated LDL is an in vitro chemically modified form of LDL and does not exist in vivo. Both acetylated LDL and oxidized LDL, are taken up by macrophages, transforming those cells into foam cells. In most cases, all components of LDL are susceptible to oxidation, producin an oxidized form of LDL (oxLDL). The uptake of oxLDL by arterial macrophages is pivotal in the formation of plaques. Unlike unmodified LDL, oxLDL is taken up by arterial wall macrophages in an unregulated manner via LDL scavenger receptors. Oxysterols are 10-100X more reactive than native cholesterol, with the most toxic of these being 7-ketocholesterol (7KC), which is also the most abundant in oxLDL.
• 7KC 7-ketocholesterol
• 7KC is a pro- inflammatory, pro-oxidant, pro-apoptotic, and fibrogenic molecule that alters endothelial cell function by disrupting cell membranes and critical ion transport pathways for vasodilatory response.
• a subject or patient at risk for, or suffering from, high cholesterol may have a 7KC concentration higher than about 109.8 nmol/L nmol/L.
• hypercholesterolemic A subject or patient suffering from, or at risk of developing high cholesterol may be referred to as hypercholesterolemic.
• hypercholesterolemia may be assessed by measuring the percentage of plasma oxysterols, for example the percentage of 7-KC in plasma oxysterols.
• 7 KC may account for about 57% of the plasma oxysterols.
• 7KC is followed by 7-a/p hydroxycholesterol (at 21% of plasma oxysterols), which is a direct product of 7KC metabolism.
• NASH nonalcoholic steatohepatitis
• Altered cholesterol homeostasis and transport contribute to the accumulation of free cholesterol in the liver, which in turn contributes to NAFLD (Non-alcoholic fatty liver disease) via damage to hepatocytes and the activation of non-parenchymal cells.
• NAFLD Non-alcoholic fatty liver disease
• the overload of free cholesterol in and around the mitochondria induces mitochondrial dysfunction and promotes inflammation, fibrosis and hepatocyte death.
• Other cholesterol-associated diseases include pulmonary alveolar proteinosis (PAP), eye disease, neurodegenerative diseases, Niemann Pick Type C (NPC), and Lysosomal Acid Lipase (LAL) deficiency.
• PAP pulmonary alveolar proteinosis
• NPC Niemann Pick Type C
• LAL Lysosomal Acid Lipase
• oxysterols and, in particular 7KC cause degeneration of retinal cells.
• increased oxysterol levels may play a role in various eye diseases including macular degeneration (AMD), choroidal neovascularization (CNV), glaucoma, and cataracts.
• Increased oxysterol levels may also result in alterations in brain cholesterol metabolism.
• Cholesterol metabolism may be an integral part of several brain disorders including Alzheimer’s disease, Amyotrophic Lateral Sclerosis (ALS), Parkinson’s disease, and dementia progression.
• ALS Amyotrophic Lateral Sclerosis
• Various oxysterols derived from the auto-oxidation of cholesterol, including 7KC have been identified in post-mortem brains of patients with Alzheimer’s disease.
• Chronic epilepsy may also share many of these pathologies. Specifically, a link has been suggested between epilepsy and atherosclerosis. Thus, treatment of atherosclerosis, such as the presently disclosed compositions, cells, and methods may lessen the effects of epilepsy.
• 7KC is highly cytotoxic to neuronal cells and has been suspected to be involved in the progression of various neurological diseases.
• oxysterols unlike cholesterol, can cross the blood brain barrier (BBB) and accumulate in brain tissue, ultimately causing neurodegeneration.
• BBB blood brain barrier
• Various other diseases may be linked with increased cholesterol levels.
• NPC Niemann Pick Type C
• patients with Niemann Pick Type C are inable to clear cholesterol, causing the accumulation of cholesterol and oxysterols in mostly the liver, spleen, and brain.
• a positive correlation between the 7KC profile and the severity of the disease has been reported.
• patients with Lysosomal Acid Lipase (LAL) deficiency accumulate cholesterol esters and triglycerides in lysosomes, and can present with hypercholesterolemia, hyperlipidemia, and/or atherosclerosis.
• LAL Lysosomal Acid Lipase
• These patients also have very high levels of oxysterols, including 7KC, in their plasma. Increased formation of oxysterols further increases oxidative stress worsens the condition.
• the disclosed compositions and methods are useful in treating diseases or conditions associated with excess cholesterol and/or fat deposits in cells, tissues, and organs.
• the disease or condition may be associated with excess cholesterol and/or the presence of one or more oxidized cholesterol species, such as 7- ketocholesterol.
• the disease or condition may be one or more of fatty liver disease, atherosclerosis, heart failure, stroke, ischemia, coronary heart disease, eye disease, neurodegenerative and neurological disease, diseases of the eye, such as macular degeneration, pulmonary dysfunction, etc.
• compositions, cells, methods, and therapies may aid in treating, reducing, or reversing various diseases, disorders, or conditions related to excess cholesterol.
• the disease, disorder, or condition may be one or more of early type II lesions (i.e. macrophage foam cell formation), type III lesions or preatheromas (i.e. having small pools of extracellular lipids), type IV lesions or atheromas (i.e. having a core of extracellular lipids), type V lesions or fibroatheromas (i.e. atheromas with fibrous thickening).
• compositions, constructs, cells, methods, therapies, and systems may aid in reducing the concentration of cholesterol in the blood of a patient.
• a decrease in the concentration of cholesterol in the blood e.g. weight per volume, such as mg/dl, or molarity, such as mmol/L
• a decrease in the concentration of cholesterol in the blood may be greater than about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, or 90% and less than about 100%, 95%, 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, or 2%.
• the decrease in cholesterol levels upon treatment may be reflected in an increase in the concentration or amount of one or more cholesterol catabolites, for example an increase of greater than 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 1.5X, 2.0X, 2.5X, 3.0X, 3.5X, 4.0X, 4.5X, 5.0X, 10X, 20X, 30X, 40X, 50X, 60X, 70X, 80X, 90X, 100X, 200X, 300X, 400X, 500X, 600X, 700X, 800X, or more, and less than about 2000X, 1500X, 1000X, 500X, 400X, 300X, 200X, 100X, 90X, 80X, 70X, 60X, 50X, 40X, 30X, 20X, 10X, 5X, 4X, 3X, 2X, 100%, 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, or 10% over the concentration of catabolite prior to treatment.
• the cell media was assayed for catabolite concentration.
• Total cellular protein was extracted using RIPA buffer and total protein concentrations were quantified using a standard BCA kit.
• FIG. 1 the expression of CDP in 293T kidney epithelial cells resulted in a ⁇ 300-fold increase catabolite concentration in culture media from baseline levels (media of cells expressing only empty plasmid i.e. no CDP). Coexpression of this fusion protein with CDP increased catabolite production ⁇ 4-fold compared to CDP alone.
• Tom20 has 50N-terminal residues embedded in the outer mitochondrial membrane (OMM) and 95 residues in the cytoplasm. Fusion of StAR residues 63-285 to the carboxyl terminus of Tom20 (i.e.
• Claim 1 A nucleic acid composition comprising: a coding sequence for Steroidogenic Acute Regulatory (StAR) protein; and a coding sequence for a cholesterol degrading protein (CDP) comprising one or more of cholesterol dehydrogenase (CholD), 3-ketosteroid A1 -dehydrogenase (A1- KstD), anoxic cholesterol metabolism B enzyme (acmB), 3-ketosteroid 9a- hydroxylase (KshAB), 3
• CDP cholesterol degrading protein
• Claim 2 The nucleic acid of claim 1 , wherein the StAR protein comprises residues 63 to 188 of SEQ ID NO:7.
• Claim 3 The nucleic acid of claim 1 or claim 2, wherein the StAR protein comprises a duplication of residues 63-188 of SEQ ID NO:7.
• Claim 4 The nucleic acid of any of claim 1 to claim 3, wherein the StAR protein comprises at least a portion of the TOMM20 protein.
• Claim 5 The nucleic acid of any of claim 1 to claim 4, wherein the CDP comprises P450.
• Claim 6 The nucleic acid of any of claim 1 to claim 5, wherein the CDP comprises P450-ferredoxin reductase-ferredoxin fusion protein (P450-FdxR-Fdx).
• Claim 7 The nucleic acid of any of claim 1 to claim 6, wherein the StAR protein and the CDP comprise at least one mitochondrial signaling protein.
• Claim 8 The nucleic acid of any of claim 1 to claim 7, wherein the nucleic acid further includes a cell-specific promoter sequence.
• Claim 9 The nucleic acid of any of claim 1 to claim 8, wherein the nucleic acid is comprised in a lipo nanoparticle.
• Claim 10 The nucleic acid of any of claim 1 to claim 8, wherein the nucleic acid is comprised in a viral vector.
• Claim 11 The nucleic acid of any of claim 1 to claim 10, for use in treating a subject having high cholesterol.
• Claim 12 The nucleic acid of any of claim 1 to claim 11 , for use in transforming a mammalian cell.
• Claim 13 The nucleic acid of claim 12, wherein the mammalian cell is transformed in-vitro.
• Claim 14 The nucleic acid of claim 12, wherein the mammalian cell is transformed in-vivo.
• Claim 15 The nucleic acid of any of claim 1 to claim 14, wherein the nucleic acid is comprised in a lipo nanoparticle.
• Claim 16 The nucleic acid of any of claim 1 to claim 15, wherein the nucleic acid is comprised in a cell of subject suffering from or at risk of developing high cholesterol.
• Claim 17 The nucleic acid of any of claim 1 to claim 16, for use in reducing cholesterol in a subject having high cholesterol.
• Claim 18 A mammalian cell comprising: a coding sequence for an engineered Steroidogenic Acute Regulatory (StAR) protein or portion thereof; and a coding sequence for a cholesterol degrading protein (CDP) comprising one or more of cholesterol dehydrogenase (CholD), 3-ketosteroid A1 -dehydrogenase (A1- KstD), anoxic cholesterol metabolism B enzyme (acmB), 3-ketosteroid 9a- hydroxylase (KshAB), 3
• StAR Steroidogenic Acute Regulatory
• CDP cholesterol degrading protein
• Claim 19 The mammalian cell of claim 18, wherein the StAR protein comprises residues 63 to 188 of SEQ ID NO:7.
• Claim 20 The nucleic acid of claim 18 or claim 19, wherein the StAR protein comprises a duplication of residues 63-188 of SEQ ID NO:7.
• Claim 21 The mammalian cell of any of claim 18 to claim 20, wherein the StAR protein comprises at least a portion of a TOMM20 protein sequence.
• Claim 22 The mammalian cell of any of claim 18 to claim 21 , wherein the CDP comprises P450.
• Claim 23 The mammalian cell of any of claim 18 to claim 22, wherein the CDP comprises P450-ferredoxin reductase-ferredoxin fusion protein (P450-FdxR-Fdx).
• Claim 24 The mammalian cell of any of claim 18 to claim 23, wherein the StAR protein and the CDP comprise at least one mitochondrial signaling protein.
• Claim 25 The mammalian cell of any of claim 18 to claim 24, wherein the coding sequence of the StAR or CDP further includes a cell-specific promoter sequence.
• Claim 26 The mammalian cell of any of claim 18 to claim 25, wherein the coding sequence of the StAR or CDP is integrated into the mammalian cell’s genome.
• Claim 27 The mammalian cell of any of claim 18 to claim 25, wherein the coding sequence of the StAR or CDP is not integrated into the genome of the mammalian cell.
• Claim 28 The mammalian cell of any of claim 18 to claim 27, for use in treating a subject suffering from or at risk of developing high cholesterol.
• Claim 29 The mammalian cell of any of claim 18 to claim 28, for use in reducing cholesterol in a subject.
• Claim 30 The mammalian cell of any of claim 18 to claim 29, wherein the mammalian cell is selected from a liver cell, neuronal cell, bone cell, muscle cell, endothelial cell, epithelial cell, immune cell, hematopoietic cell, and stem cell or precursor cell thereof.
• Claim 31 The mammalian cell of any of claim 18 to claim 29, wherein the mammalian cell is selected from a liver cell, neuronal cell, bone cell, muscle cell, endothelial cell, epithelial cell, immune cell, hematopoietic cell, and stem cell or precursor cell thereof.
• a mammalian cell comprising: an engineered Steroidogenic Acute Regulatory (StAR) protein or portion thereof; and a cholesterol degrading protein (CDP) comprising one or more of cholesterol dehydrogenase (CholD), 3-ketosteroid A1 -dehydrogenase (A1-KstD), anoxic cholesterol metabolism B enzyme (acmB), 3-ketosteroid 9a-hydroxylase (KshAB), 3
• CDP cholesterol degrading protein
• Claim 32 The mammalian cell of claim 31 , wherein the StAR protein comprises residues 63 to 188 of SEQ ID NO:7.
• Claim 33 The mammalian cell of claim 31 or claim 32, wherein the StAR protein comprises a duplication of residues 63-188 of SEQ ID NO:7.
• Claim 34 The mammalian cell of any of claim 31 to claim 33, wherein the StAR protein comprises at least a portion of the TOMM20 protein.
• Claim 35 The mammalian cell of any of claim 31 to claim 34, wherein the CDP comprises P450.
• Claim 36 The mammalian cell of any of claim 31 to claim 35, wherein the CDP comprises P450-ferredoxin reductase-ferredoxin fusion protein (P450-FdxR-Fdx).
• Claim 37 The mammalian cell of any of claim 31 to claim 36, wherein the StAR protein and the CDP comprise at least one mitochondrial signaling protein.
• Claim 38 The mammalian cell of any of claim 31 to claim 37, wherein at least 10% of the total amount of StAR protein and CDP in the cell are located in a mitochondrial membrane.
• Claim 39 The mammalian cell of any of claim 31 to claim 38, wherein the StAR protein or the CDP is coded for by a gene integrated into the genome of the mammalian cell.
• Claim 40 The mammalian cell of any of claim 31 to claim 39, wherein the StAR protein or the CDP is coded for by an extra genomic gene.
• Claim 41 The mammalian cell of any of claim 31 to claim 39, for use in treating a subject suffering from or at risk of developing high cholesterol.
• Claim 42 The mammalian cell of any of claim 31 to claim 40, for use in reducing cholesterol in a subject.
• Claim 43 The mammalian cell of any of claim 18 to claim 29, wherein the cell is selected from a liver cell, neuronal cell, bone cell, muscle cell, endothelial cell, epithelial cell, immune cell, hematopoietic cell, and stem cell or precursor cell thereof.
• Claim 44 The mammalian cell of any of claim 18 to claim 29, wherein the cell is selected from a liver cell, neuronal cell, bone cell, muscle cell, endothelial cell, epithelial cell, immune cell, hematopoietic cell, and stem cell or precursor cell thereof.
• a method of enhancing cholesterol degradation in a mammalian cell comprising: expressing non-native engineered Steroidogenic Acute Regulatory (StAR) protein or portion thereof; and expressing one or more of a cholesterol degrading protein (CDP) comprising cholesterol dehydrogenase (CholD), 3-ketosteroid A1 -dehydrogenase (A1-KstD), anoxic cholesterol metabolism B enzyme (acmB), 3-ketosteroid 9a-hydroxylase (KshAB), 3
• CDP cholesterol degrading protein
• CholD cholesterol dehydrogenase
• A1-KstD 3-ketosteroid A1 -dehydrogenase
• acmB anoxic cholesterol metabolism B enzyme
• KshAB 3-ketosteroid 9a-
• Claim 45 The method of claim 44, wherein the StAR protein comprises residues 63 to 188 of SEQ ID NO:7.
• Claim 46 The method of claim 44 or claim 45, wherein the StAR protein comprises a duplication of residues 63-188 of SEQ ID NO:7.
• Claim 47 The method of any of claim 44 to claim 46, wherein the StAR protein comprises at least a portion of the TOMM20 protein.
• Claim 48 The method of any of claim 44 to claim 47, wherein the CDP comprises P450.
• Claim 49 The method of any of claim 44 to claim 48, wherein the CDP comprises P450-ferredoxin reductase-ferredoxin fusion protein (P450-FdxR-Fdx).
• Claim 50 The method of any of claim 44 to claim 49, wherein the StAR protein and the CDP comprise at least one mitochondrial signaling protein.
• Claim 51 The method of any of claim 44 to claim 50, wherein the StAR protein and CDP are expressed from a cell-specific promoter sequence.
• Claim 52 The method of any of claim 44 to claim 51 , for use in treating a subject suffering from or at risk of developing high cholesterol.
• Claim 53 The method of any of claim 44 to claim 52, for use in reducing cholesterol in a subject having high cholesterol.
• Claim 54 The method of any of claim 44 to claim 53, wherein at least about 10% of the total amount of StAR protein and CDP in the cell is located in a mitochondrial membrane.
• Claim 55 The method of any of claim 44 to claim 54, wherein the StAR protein or the CDP is expressed from a gene integrated into the mammalian cell’s genome.
• Claim 56 The method of any of claim 44 to claim 54, wherein the StAR protein or the CDP is from an extra genomic gene.
• Claim 57 The method of any of claim 44 to claim 56, for use in treating a subject suffering from or at risk of developing high cholesterol.
• Claim 58 The method of any of claim 44 to claim 57, for use in reducing cholesterol in a subject.
• Claim 59 The method of any of claim 44 to claim 58, wherein the cell is selected from a liver cell, neuronal cell, bone cell, muscle cell, endothelial cell, epithelial cell, immune cell, hematopoietic cell, and stem cell or precursor cell thereof.
• Claim 60 The method of any of claim 44 to claim 59, wherein the cholesterol is LDL.
• Claim 61 The method of any of claim 44 to claim 59, wherein the cholesterol is 7KC.
• Claim 62 A method of treating a patient at risk of developing or suffering from high cholesterol comprising: administering to the patient at least one modified cell, the modified cell comprising a non-native engineered Steroidogenic Acute Regulatory (StAR) protein or portion thereof; and one or more of a cholesterol degrading protein (CDP) comprising cholesterol dehydrogenase (CholD), 3-ketosteroid A1 -dehydrogenase (A1-KstD), anoxic cholesterol metabolism B enzyme (acmB), 3-ketosteroid 9a-hydroxylase (KshAB), 3
• CDP cholesterol degrading protein
• CholD cholesterol dehydrogenase
• A1-KstD 3-ketosteroid A1 -dehydrogenase
• acmB anoxi
• Claim 63 The method of claim 62, wherein the StAR protein comprises residues 63 to 188 of SEQ ID NO:7.
• Claim 64 The method of claim 62 or claim 63, wherein the StAR protein comprises a duplication of residues 63-188 of SEQ ID NO:7.
• Claim 65 The method of claim 62 to claim 64, wherein the StAR protein comprises at least a portion of the TOMM20 protein.
• Claim 66 The method of claim 62 to claim 65, wherein the CDP comprises P450.
• Claim 67 The method of claim 62 to claim 66, wherein the CDP comprises P450-ferredoxin reductase-ferredoxin fusion protein (P450-FdxR-Fdx).
• Claim 68 The method of claim 62 to claim 67, wherein the StAR protein and the CDP comprise at least one mitochondrial signaling protein.
• Claim 69 The method of claim 62 to claim 68, wherein the StAR protein and CDP are expressed from a cell-specific promoter sequence.
• Claim 70 The method of claim 62 to claim 76, for use in reducing cholesterol in a subject having high cholesterol.
• Claim 71 The method of claim 62 to claim 71 , wherein at least about 10% of the total amount of StAR protein and CDP in the cell is located in a mitochondrial membrane.
• Claim 72 The method of claim 62 to claim 72, wherein the StAR protein or the CDP is expressed from a gene integrated into the mammalian cell’s genome.
• Claim 73 The method of claim 62 to claim 72, wherein the StAR protein or the CDP is from an extra genomic gene.
• Claim 74 The method of claim 62 to claim 73, wherein the patient has a total cholesterol of greater than 200 mg/dl.
• Claim 75 The method of claim 62 to claim 74, wherein the patient suffers from non-alcoholic fatty liver disease.
• Claim 76 The method of claim 62 to claim 74, wherein the patient suffers from non-alcoholic steatohepatitis.
• Claim 77 The method of claim 62 to claim 76, wherein the modified cell is derived from a liver cell, neuronal cell, bone cell, muscle cell, endothelial cell, epithelial cell, immune cell, hematopoietic cell, and stem cell or precursor cell thereof.
• Claim 78 The method of claim 62 to claim 77, wherein the method reduces the total cholesterol concentration in the patient's blood by greater than 5%.
• Claim 79 A method of modifying a mammalian mitochondrion comprising: inserting at least one non-native engineered Steroidogenic Acute Regulatory (StAR) protein or portion thereof into the mammalian mitochondrion outer membrane; and wherein the mitochondrion comprises one or more of a cholesterol degrading protein (CDP) comprising cholesterol dehydrogenase (CholD), 3-ketosteroid A1- dehydrogenase (A1-KstD), anoxic cholesterol metabolism B enzyme (acmB), 3- ketosteroid 9a-hydroxylase (KshAB), 3
• CDP cholesterol degrading protein
• CholD cholesterol dehydrogenase
• A1-KstD 3-ketosteroid A1- dehydrogenase
• Claim 80 The method of claim 79, wherein the StAR protein comprises residues 63 to 188 of SEQ ID NO:7.
• Claim 81 The method of claim 79 or claim 80, wherein the StAR protein comprises a duplication of residues 63-188 of SEQ ID NO:7.
• Claim 82 The method of any of claim 79 to claim 81 , wherein the StAR protein comprises at least a portion of the TOMM20 protein.
• Claim 83 The method of any of claim 79 to claim 82, wherein the CDP comprises P450.
• Claim 84 The method of any of claim 79 to claim 83, wherein the CDP comprises P450-ferredoxin reductase-ferredoxin fusion protein (P450-FdxR-Fdx).
• Claim 85 The method of any of claim 79 to claim 84, wherein the StAR protein and the CDP comprise at least one mitochondrial signaling protein.
• Claim 86 The method of any of claim 79 to claim 85, wherein the StAR protein and CDP are expressed from a cell-specific promoter sequence.
• Claim 87 The method of any of claim 79 to claim 86, for use in treating a subject suffering from or at risk of developing high cholesterol.
• Claim 88 The method of any of claim 79 to claim 87, for use in reducing cholesterol in a subject having high cholesterol.
• Claim 89 The method of any of claim 79 to claim 88, wherein at least about 10% of the total amount of StAR protein and CDP in the cell is located in a mitochondrial membrane.
• Claim 90 The method of any of claim 79 to claim 90, wherein the StAR protein or the CDP is expressed from a gene integrated into the mammalian cell’s genome.
• Claim 91 The method of any of claim 79 to claim 89, wherein the StAR protein or the CDP is from an extra genomic gene.
• Claim 92 The method of any of claim 79 to claim 91 , for use in treating a subject suffering from or at risk of developing high cholesterol.
• Claim 93 The method of any of claim 79 to claim 92, for use in reducing cholesterol in a subject.
• Claim 94 The method of any of claim 79 to claim 93, wherein the cell is selected from a liver cell, neuronal cell, bone cell, muscle cell, endothelial cell, epithelial cell, immune cell, hematopoietic cell, and stem cell or precursor cell thereof.

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